www.elsevier.com/locate/brainres

Brain Research 1057

Research Report

Robust axonal sprouting and synaptogenesis in organotypic slice cultures

of rat cerebellum exposed to increased potassium chloride

Suzanne Chena,b, Kasunari Hiratab, Yuqin Rena,b, Mutsuyuki Sugimorib,

Rodolfo Llinasb, Dean E. Hillmana,b,*

aDepartment of Otolaryngology and Physiology and Neuroscience, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USAbDepartment of Physiology/Neuroscience, New York University School of Medicine, New York, NY 10016, USA

Accepted 19 July 2005

Available online 25 August 2005

Abstract

Organotypic slices of the rat cerebellum, cultured in physiological levels [K+]o (5 mM) for 14 days, loose the majority of granule cells in

the anterior lobe resulting in few axons and atypical Purkinje cell dendrites with vacant spines. When the culture medium was switched from

5 mM to 20, 30 or 40 mM [K+]o during the last 7 days of cultures, slices developed axons with numerous vesicle-filled boutons that made

synaptic contact with Purkinje cell spines. Most boutons had one or two spine profile contacts, while some were unusually large. Enlarged

boutons abutted Purkinje cell somata or their dendrites, causing intervening spines to invaginate terminals to form rosette synaptic

complexes. Calbindin immuno-labeling excluded Purkinje cell axonal collaterals as the source of rosette boutons and suggested a granule cell

origin. Quantification of vacant spines as compared to those on boutons revealed a threshold for potassium, between 10 and 20 mM, where

the number of synaptic spines increased and vacant spines decreased drastically. These findings suggest that elevated [K+]o triggers an

activity-dependent plasticity in rat cerebellar slice cultures by promoting axonal sprouting with formation of vesicle-filled boutons and

synaptogenesis on open receptor sites of Purkinje cell spines.

D 2005 Elsevier B.V. All rights reserved.

Theme: Development and regeneration

Topic: Formation and specificity of synapses

Keywords: Axogenesis; Synaptic vesicle production; Enlarged bouton; Granule cell; In vitro method; Purkinje cell; Rosette synaptic complexes; Vacant spine

1. Introduction

Organotypic cultures from various brain regions retain

much of the local morphological framework but show

changes in connectivity suggesting circuit plasticity in vitro

[4,11,39,49]. In rat cerebellar slice cultures using standard

media, there is a substantial loss of granule cells (GCs) and

their axons, thus removing a major input to Purkinje cells

(PCs) as well as to inhibitory interneurons. Because PCs in

organ culture are very resistant to cell death [1,17,21], the

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.07.039

* Corresponding author. Department of Otolaryngology and Physiology

and Neuroscience, New York University School of Medicine, 550 First

Avenue, New York, NY 10016, USA.

E-mail address: hillmd01@endeavor.med.nyu.edu (D.E. Hillman).

neuropil is composed mostly of PC dendrites that are

studded with vacant spines (spines without presynaptic

contact) and a few spine synapses with PC axon collaterals

[42,43]. Physiological analysis showed that these neurons

have the expected resting and spike potential properties but

slowed membrane repolarization and a lack of spontaneous

synaptic activity [16,32].

While using high [K+]o (30 mM) in slice culture media to

investigate neurotoxicity of PCs [28,29], we were surprised

to find a neuropil with spine synapses and enlarged

presynaptic boutons, instead of numerous vacant spines as

observed in standard 5 mM [K+]o [12]. Previous studies

reported that membrane depolarization by elevated [K+]oreleased transmitter [14,51] as well as enhanced GC survival

in dissociated cell cultures (DCC) [5,19,20]. Here, we report

(2005) 88 – 97

S. Chen et al. / Brain Research 1057 (2005) 88–97 89

that [K+]o treatment engenders plasticity in circuitry by

axonal sprouting, formation of vesicle-filled boutons and

synaptogenesis yielding PC spine rosette complexes that are

not observed in the normal cerebellar cortex.

2. Materials and methods

The animal protocol utilized here was approved by

Institutional Animal Care and Use Committee at New York

University School of Medicine and followed the guidelines

issued by the National Institutes of Health. Sprague–

Dawley rat pups, accompanied by their mother, were

purchased from Taconic (Germantown, NY).

For in situ fixation, 12-day-old (P12) pups were

anesthetized with pentobarbital (40 mg/kg body weight,

i.p.) and then perfused through the heart with a wash

solution of 0.2 M phosphate buffer (PB) followed by a

fixative consisting of 1% paraformaldehyde and 1%

glutaraldehyde in 0.12 M PB. Midsagittal sections of the

cerebellum were made with a vibratome at 50 Am.

For slice cultures, P12 pups were anesthetized, and their

brains were aseptically removed and immersed in cold

sterile artificial CSF-like ringer [29]. The vermis of the

cerebellum was sectioned sagittally into 300 Am slices using

a Microslicer (Dosaka EM, Japan). Each slice was collected

in the CSF ringer and placed on a porous membrane insert

(Millicell CM, Millipore, MA) at the interface between air

and a standard culture medium. It consists of 50% Minimum

Essential Medium, 25% Earl’s Balanced Salt Solution

(EBSS), 25% heat-inactivated horse serum, 1 mM l-

glutamine and 36 mM d-glucose. All slices were incubated

at 35 -C in a humidified incubator with 7% CO2 (pH 7.3)

and fed every 2 or 3 day [46].

Control slices were cultured in standard medium (5 mM

KCl) for 14 days, whereas experimental slices were cultured

in the standard medium for 7 days and then switched to 10,

20, 30 or 40 mM [K+]o for 7 more days. This 7-day

preconditioning in 5 mM [K+]o stabilized the neuronal

population following marked reduction in the number of

GCs and a complete degeneration of parallel fibers.

At 14 days in vitro (14 DIV), slices of all groups were

fixed for 2 h, in 1% paraformaldehyde and 1% glutaraldehyde

in EBSS for ultrastructure quantification. They were post-

fixed in 1% OsO4 containing 1% potassium ferrocyanide for

1 h and left in 2% aqueous uranyl acetate overnight. Slices

were dehydrated gradually from 70% to 100% ethyl alcohol

and embedded in Durcupan (Sigma/Fluka, St. Louis, MO).

Ultrathin sections were cut and collected on Formvar-coated

grids. They were double stained with uranyl acetate and lead

citrate for examination in a JEOL 100C electron microscope.

Calbindin immuno-EM was used to determine the extent

of PC axon collaterals as a bouton source. Slices from all

groups were fixed in 4% paraformaldehyde and 0.15%

glutaraldehyde in EBSS for 2 h. Slices were immersed for 1

h in 5% normal goat serum with 1% bovine serum albumin

(minimizes background) and then reacted with monoclonal

calbindin antibodies (1:10,000 dilution, Swant, Switzerland)

overnight at 4 -C. On the following day, the slices were

incubated with a secondary antibody and then processed

using an ABC Elite Kit (Vector Lab, Burlingame, CA) and

DAB as the chromagen. The reacted slices were washed

several times with 0.1 M phosphate buffer (PB) and then

postfixed in 1% OsO4 in pH 8.0 PB for 30 min. Slices were

embedded in Durcupan, and ultrathin sections were pro-

cessed as described above. Some slices were counterstained

with cresyl violet (Nissl) before alcohol dehydration and

coverslipped with Permount for LM.

Ultrastructural quantification of spines and boutons was

done to assess the effect of [K+]o on bouton development

and synaptogenesis. Due to an anterior–posterior difference

in the number of surviving GCs and synapses (preliminary

report [13]), we used only lobules I–IV which consistently

loose the majority of GCs. Furthermore, areas exhibiting a

distinct molecular layer were sampled and photographed at

6600� for each [K+]o group. The EM films were digitally

scanned and printed using PhotoShop 5.0 software (Adobe,

San Jose, CA).

Spines in section profiles were identified at high

magnification using the zoom-in tool in Photoshop. Spine

necks in cross-section typically have one ER profile in the

center of the projection (see Figs. 1D–F). The identification

between spines and parallel fibers was possible by the single

ER profile in the spine neck and microtubules in parallel

fibers. Each quantified spine was characterized as being a

spine by its expanded head and one or more ER folds.

Active zones were not considered a criterion. Normally, in

vivo, one, and on occasion two, PC spine makes synaptic

contacts with each parallel fiber bouton. In control slices,

PC spines without a presynaptic partner were predominant,

while, in experimental preparations, multiple spines (three

or more) frequently contacted an enlarged bouton. These

spine/bouton relationships provided a natural classification

for four categories: 1) vacant- (without a presynaptic

bouton), 2) single-, 3) dual- or 4) multiple-spines per

bouton. For each category, the number of spine heads

appearing in a micrograph was recorded together with the

total number of spines on each film (approximately, 100

Am2). The mean and standard deviation for each spine/

bouton type were determined together with percent differ-

ences between control and experimental groups (Table 1).

Statistical significance was calculated using Student’s t test.

3. Results

3.1. P12-in-situ-fixed sagittal sections

Nissl-stained sagittal sections of P12 displayed a thick

external granule layer, a distinct molecular layer and

numerous GCs below a single layer of PCs (Fig. 1A).

Ultrastructural analysis of the molecular layer in the anterior

Fig. 1. LM and EM images of P12, 5 mM and 30 mM KCl slices at DIV 14. (A) Light micrograph of P12-in-situ-fixed Nissl-stained section displays a thick

external granule layer (EGL), a distinct molecular layer (ML), a single PC layer (PCL) and numerous GCs below the PCL. (B) EM analysis of the neuropil at

P12 revealed numerous parallel fibers (Pf) and PC dendrites (PCD) with primitive spines having growth cone appearances (s*) in synaptic contacts (arrows)

with parallel fiber boutons. (C) 1-Am sections of control (5 mM KCl) slices that were stained with toluidine blue show a cerebellar cortex with distinct laminae:

molecular (ML), PC, GC layers as well as a white matter (WM). Note that PC dendrites (arrows) in the ML run parallel to the pial surface. (D) EM analysis of

the neuropil from 5 mM [K+]o consists mostly of vacant spines without parallel fibers or boutons. Note that there is a striking difference in the neuropil

composition as compared with that of P12. (E) A bouton (B*), likely from stellate interneurons, has a direct apposition to a medium-sized PC dendrite as is

seen, typically, in vivo. (1F) A 30 mM [K+]o preparation has numerous parallel fibers (Pf) and boutons (B) forming synaptic contacts with PC spines. The spine

neck can be identified by a narrow tube of ER that extends into the spine head where it elaborates into the spine apparatus (D–F, arrowheads). Scale bar equals

to 10 Am (A and C) and 1 Am (B, D–F).

S. Chen et al. / Brain Research 1057 (2005) 88–9790

lobe revealed masses of parallel fibers crossing through PC

dendritic branches, but only a few boutons appeared in the

field. Primitive spines without narrowing of the neck

protruded from the PC dendrites to contact one or two

small boutons of parallel fibers (Fig. 1B).

3.2. 14 DIV control slices (5 mM KCl)

Toluidine-blue-stained 1-Am plastic sections from the

anterior lobe of control slices displayed three laminae,

i.e., molecular, PC and GC layers (Fig. 1C). In these

slices, the majority of GCs that were present in P12

donor tissue were lost (compare to Fig. 1A). The

narrowed GC layer stained faintly and was composed of

a few GCs along with astrocytes and scattered Golgi cell

somata. The external granular layer was absent (Fig. 1C).

The PC somata aligned in a single layer, but their

dendritic arbors were flattened and projected almost

parallel to the PC layer rather than toward the molecular

layer surface (Fig. 1C).

Table 1

Effect of high [K+]o on synaptogenesis in the anterior lobe

K5-A K10-A K20-A K30-A K40-A

No. of films 21 16 13 10 30

Vacant spine type, M T SD 67.1 T 21.0 73.0 T 26.2 20.0 T 17.3 8.3 T 4.5 4.33 T 2.4

% change from control +9% �70% �88% �94%

P value and significant level ns <0.001*** <0.001*** <0.001***

Mono-spine type, M T SD 5.8 T 3.0 4.3 T 3.0 10.1 T 6.0 6.6 T 2.7 2.0 T 1.5

% change from control �26% +74% +13% �66%

P value and significant level ns <0.01** <0.05* <0.001***

Dual spines type, M T SD 1.5 T 0.9 1.9 T 0.8 3.0 T 2.4 3.0 T 2.4 1.8 T 0.9

% change from control �33% +100% +100% +20%

P value and significant level ns ns ns ns

Multiple spines type, M T SD 0.57 T 1.2 0.38 T 1.0 5.0 T 7.4 3.5 T 3.3 5.6 T 7.0

% change from control �30% +780% +510% +880%

P value and significant level ns <0.001*** <0.001*** <0.001***

K5-A: 5 mM KCl from the anterior lobe, M T SD: mean T standard deviation, ns: not significant.

* P < 0.05.

** P < 0.01.

*** Very significant P < 0.001.

S. Chen et al. / Brain Research 1057 (2005) 88–97 91

Ultrastructural analysis of control slices revealed very

few parallel fibers and bouton profiles. Nevertheless,

various-sized PC dendrites with elongated small spines

Fig. 2. EM micrographs of 30 mM KCl-treated slices at 14 DIV. (A) Boutons (B)

scattered at sites along the parallel fiber (arrowheads). (C and D) Enlarged bouton

without interposed glial projections. Scale bar equals to 1 Am (B–D) and 0.5 Am

formed the bulk of the molecular layer (Fig. 1D). These

vacant spines invaginated the surface of surrounding

astrocytic projections. Occasional isolated boutons were

are seen in continuation with parallel fibers. (B) A few synaptic vesicles are

s (B) contacted the plasma membranes of PC dendrites and somata directly

(A).

S. Chen et al. / Brain Research 1057 (2005) 88–9792

opposed to the plasma membrane of PC dendrites without

spine contact. These boutons appeared to be inhibitory

terminals from stellate neurons that are seen normally in

vivo (Fig. 1E).

3.3. 14 DIV experimental slices (7-day exposure to 10, 20,

30 or 40 mM KCl)

In LM analysis, the molecular and GC layers were

similar between control and all experimental groups.

Nonetheless, the Nissl staining of GCs appeared stronger

when the KCl was increased to 20, 30 or 40 mM as

compared to control or 10 mM groups.

Ultrastructural analysis of the molecular layer in the 10

mM group was indistinguishable from control prepara-

tions. In contrast, preparations from the 20, 30 or 40 mM

[K+]o groups all had a neuropil composed of large and

Fig. 3. EM micrographs from 40 mM KCl-treated slices show enlarged vesicle-fill

presence of PC dendrites (D) and parallel fibers in the neuropil. Enlarged boutons

made direct contact on the surface of dendrites, and these boutons were larger in di

than 20 PC spines in this sectional plane. Scale bar equals to 1 Am (A–C).

small dendrites separated by axons, boutons and spines.

Small axonal bundles were oriented in various directions

(Fig. 1F). In fortunate sections, these axons were seen

continuing into vesicle-filled boutons (Fig. 2A). Further-

more, scattered or clustered synaptic vesicles were found,

atypically, in axonal shafts (Fig. 2B). The density of

synaptic vesicles was essentially the same in large or

small boutons.

3.3.1. Bouton/spine synapses

Many of the presynaptic boutons contacted one or more

PC spines. These spine heads were larger in diameter than

those of vacant spines and contained a prominent spine

apparatus that was connected by a single central ER

projection through a narrow spine neck (Figs. 1D–F,

arrowheads). At spine/bouton appositions, distinct active

zones were apposed to presynaptic vesicles.

ed boutons making synaptic contact with numerous PC spines. (A) Note the

(B) had many PC spines invaginating the bouton surface. (B) Some boutons

ameter than the postsynaptic dendrite. (C) A giant bouton (B) contacts more

Fig. 4. Calbindin immuno-EM of slices treated with 30 mM KCl (A) revealed calbindin-IR in PC spines and dendrite (D), while an enlarged bouton (B) showed

no IR. (B) Calbindin-IR in PC dendrite and opposed bouton implies that this labeled bouton is derived from a PC collateral axon and synapses on a PC

dendrite. Scale bar equals to 0.5 Am (A and B).

S. Chen et al. / Brain Research 1057 (2005) 88–97 93

3.3.2. Rosette synaptic complexes

Following 7-day exposure to 20 to 40 mM [K+]o,

rosette complexes with multiple spine contacts appeared

among normal sized boutons. The number of spines and

the amount of synaptic vesicles increased, proportionally,

with the size of rosette complexes. Most often, profiles

of these large boutons were apposed to PC dendrites

(Fig. 2C) or somata (Fig. 2D) without interposed

Fig. 5. Histogram distribution of four spine/bouton types sampled from the anterio

are vacant along with a very few mono-spine/bouton type in control and 10 mM

spines and a progressive increase in synaptic spines with each increment of [K+]o f

types toward the right bins and also a marked increase in multiple spines/bouton i

0.001, **P < 0.01 or *P < 0.05.

astrocytic glia. Remarkably, enlarged boutons exceeded

PC dendritic diameter and wrapped the dendritic surface

(Figs. 3A and B). Numerous spine heads were sand-

wiched within the apposed area and invaginated the

bouton surface (Figs. 2 and 3). Up to 20 spine heads

could be observed in synaptic contact on a single

enlarged bouton, forming rosette synaptic complexes

(Fig. 3C).

r lobe of control, 10, 20, 30 and 40 mM KCl groups. The majority of spines

KCl groups. In contrast, there is a large reduction in the number of vacant

rom 20 to 40 mM. Note a shift in the frequency distribution of spine/bouton

n the 40 mM KCl group. The significance levels are represented as ***P <

S. Chen et al. / Brain Research 1057 (2005) 88–9794

3.4. EM calbindin immunoreactivity for PC axon

identification

Enlarged boutons failed to show calbindin immunoreac-

tivity (IR) (Fig. 4A). PC dendrites, spines and axons had

strong calbindin-IR (Figs. 4A and B). Calbindin-IR boutons

of PC recurrent axon collaterals contacted IR positive PC

dendrites and somata, directly (Fig. 4B).

3.5. Spine/bouton quantification and data analysis

Quantification of four spine/bouton types in the anterior

lobe showed no difference between the control and 10 mM

[K+]o groups (Table 1). Remarkable, the number of vacant

spines was reduced by 70% in 20 mM, 88% in 30 mM and

94% in 40 mM [K+]o groups as compared to the control

(Table 1). In parallel, 20–40 mM [K+]o groups had a 4- to 8-

fold increase in the number of spines that formed multiple

contacts on large boutons. Both comparisons were highly

significant (P < 0.001). Although the number of mono- and

dual-spine/bouton types appeared to increase, the exper-

imental group differences from control were not statistically

significant (see Table 1). The histogram (Fig. 5) illustrates

similar distribution patterns between control and 10 mM

[K+]o for vacant spines and types of spine contacting

boutons. In contrast, the 20 mM [K+]o or higher groups had

sharp reductions in vacant spine number. Furthermore, with

each raise in [K+]o, there was a progressive increase in

bouton size and number of spine synapses.

4. Discussion

Numerous in vitro studies have applied various compo-

sitions of culture media to investigate neuronal survival

[2,3,38] and synaptic plasticity [18,47]. Increased [K+]olevels have been shown to release glutamate transmitter from

GCs of DCCs [14,51] and induce cell death by over-

stimulation [22,48].

Our finding shows that elevated [K+]o enhances neuronal

connectivity in cerebellar slice cultures by inducing axonal

sprouting, synaptic vesicle production and synaptogenesis.

GC survival did not have any noticeable improvement in

our delayed [K+]o treatment model, however, these neurons

do survive at near normal number when the [K+]oconcentration was increased on the first day rather than

after 7 days (our preliminary result [27]).

4.1. Axonal growth and rosette formation

Quantitative data revealed extensive rosette formations in

20–40 mM [K+]o groups in contrast to few in standard and

10 mM [K+]o. The dose response of [K+]o is seen by the

distinct shift in histogram bins from vacant spines in 5 or 10

mM [K+]o preparations to bins of increasing number of

spines on boutons with each increment of [K+]o between 20

and 40 mM (see Fig. 5). Secondly, there was a threshold for

the [K+]o response that occurred between 10 and 20 mM,

where newly formed boutons begin to make robust synapses

on PC spines.

When sagittal slices of the rat are cultured in standard

[K+]o media, the molecular layer of anterior lobes resembles

the agranular cerebellum of various models [44]. This may

be because all parallel fibers were transected and incapable

of regenerating in standard culture media. Nevertheless,

after the initial 7 days in 5 mM [K+]o and then treatment for

7 days with 20 to 40 mM [K+]o, the circuitry was rescued

through axonal sprouting by the few remaining GCs.

Frequently, small clusters of vesicles were observed along

axonal shafts in regions without boutons. Elongation of axons

provides a route for newly generated vesicles to reach target

sites on PC spines where rosette complexes could form.

These axons generated various-sized boutons filled with

synaptic vesicles that targeted vacant spines. In 20–40 mM

KCl slices, enlarged boutons were frequently opposed to the

plasmalemma of PC dendrites and soma. The interposed

spines invaginated the bouton within the opposition. Since

more than 20 spines could be found on a rosette synaptic

formation of each section profile, the total number of spine

heads per rosette could be several times larger in three

dimensions.

4.2. GC axons are likely the source of increased number of

boutons

In cultures, there is a total absence of cerebellar

afferents, and therefore any axonal terminals must be of

local origin. In our study, calbindin-IR was used as a

specific marker for PC recurrent collateral axons. We

found calbindin-IR boutons, synapsing directly on PC

dendrites and somata. However, enlarged boutons that

formed multiple synaptic contacts on PC spines lacked

calbindin-IR. Furthermore, these boutons contained round

vesicles, rather than irregular and flattened vesicles, and

occasionally were observed in continuity with parallel

fiber-like axons. These results suggest that GC axons are

the source of reactive boutons.

Other studies in explant cultures show sprouting of PC

recurrent axon collaterals and formation of inhibitory

synapses on PC somata as well as, heterotypically, on PC

spines when there is a deficit in the number of GCs

[16,41,43]. In roller-tube organ cultures, large boutons

resembling mossy fibers were suggested to arise from deep

cerebellar nuclei and make synaptic contact on dendrites of

granule and Golgi cells, as well as on PC spines [31]. In

mutant mice having granuloprival cerebella, large presy-

naptic boutons were proposed to be from both climbing and

mossy fibers [34]. More recently, hyperspiny PCs and

nodding mutants were found to have large boutons

contacted by multiple PC spines. Sotelo suggested that

these axons arise from GCs in both mutants as well as from

stellate axonal terminals in nodding mice [44].

S. Chen et al. / Brain Research 1057 (2005) 88–97 95

4.3. Neuronal survival

Elevation of [K+]o from normal physiological levels of

3–5 mM up to 25 mM in DCCs was reported to improve

GC survival of rat cerebella [5,19,20]. The same level of GC

survival in DCCs could be achieved by glutaminergic

agonists [5–7,24,30]. In our organotypic slices, both control

and 10 mM [K+]o groups had good survival of PCs but

exhibited only a narrow band of GC layer throughout the

cerebellar vermis except in lobules 9 and 10 (preliminary

report [13]). Following 7-day exposure to 20, 30 or 40 mM

[K+]o, the GC layer remained thin but had an increased

intensity of Nissl staining. This finding indicates that the

few remaining GCs acquired ribosomes (indicated by

increased Nissl staining) for protein synthesis serving

neurite outgrowth and synaptic vesicle production.

In DCCs of the mouse, GCs flourish equally well in

either low or high [K+]o [35–37]. This mouse to rat species

difference has been attributed to slower AMPA receptor

desensitization in mice allowing more Ca++ entry to sustain

the mouse GCs [35]. Likewise, we found no massive GC

loss in slice cultures of the mouse using our standard

medium (unpublished data). The apparent inability of rat

GCs to sustain themselves in lobules 1–8 following trans-

section of parallel fibers raises important questions on

conditions controlling the survival of specific neuronal types

in vitro.

There was increased risk for PC excitotoxicity in 20–40

mM KCl. Hirata et al. reported a 30% reduction in PC

number of slice cultures at 30 mM KCl as compared to

standard media [28,29]. Concurrently, the remaining PC

dendritic trees were shorter and there were fewer branch

points. Ultrastructural study confirmed PC atrophy showing

pathological changes in ER and mitochondria, with the most

severe alterations occurring at 40 mM KCl.

4.4. Mechanism of circuitry enhancement by increased

[K+]o

Presumably, there is a chronic membrane depolarization

by 20–40 mM [K+]o that opens voltage-gated calcium

channels [10,14,20,33] and, possibly, even ligand-depend-

ent NMDA types [50]. This calcium flux is believed to

activate signal transduction cascades [19,23] leading to de

novo protein synthesis [10,20,33]. The importance of Ca++

for neuronal survival is supported by the failure of enhanced

GC survival following the depolarizing agent, veratridine,

which acts on sodium channels without Ca++ influx [15].

Such plasticity is consistent with studies showing that

chronic electrical stimulation of denervated cortex produces

significant axonal re-growth and circuit remodeling [40].

Remarkably, static electric fields facilitate axonal growth

and apparently restore some function in the adult spinal cord

[8,9].

The availability of PC spines appears to play a key role in

robust synaptogenesis leading to formation of rosette

boutons. Since the number of presynaptic boutons was

inadequate for postsynaptic targets, multiple PC spines

contacted boutons and thus formed rosette complexes. One

reason for this disproportional relationship is a constancy

principle for the total contact area of active zones that

sustains large numbers of spines following deafferentation

[25,26]. Regardless of increases or decreases in the number

of afferent fibers, total contact area of postsynaptic densities

on each PC remains constant [25,26]. These findings

suggest that individual postsynaptic areas shift dynamically

between contact sites facilitating plastic changes such as the

formation of rosette synaptic complexes found in this study.

The constancy of postsynaptic contacts is supported by

studies showing proliferation of spines over the entire PC

dendritic arbor that follows climbing fiber deafferentation

[45] and in agranular cerebella where spines elongate to

reach available presynaptic sites when preterminals are

scarce [34].

5. Conclusions

This study shows that rat cerebellar slices, cultured with

3–8� higher [K+]o, have marked axonal sprouting, synaptic

vesicle production, rosette bouton formations and synapto-

genesis. The results suggest that the elevated K+ environ-

ment is responsible for an activity-dependent plasticity of

neuronal circuitry that may have implications for study of

stroke and spinal cord injury in the adult.

Acknowledgments

This work was supported by NIH-NINCDS NS-13742

and NYS Spinal Cord Injury Research Program. We thank

Dr. Eric Lang for his critical reading with the manuscript.

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